Diagnosis, prevention and treatment of Crohn&#39;s disease using the OmpC antigen

ABSTRACT

The present invention provides a method of diagnosing Crohn&#39;s disease in a subject by determining the presence or absence of IgA anti-OmpC antibodies in the subject, where the presence of the IgA anti-OmpC antibodies indicates that the subject has Crohn&#39;s disease.

This application was made with government support under USPHS grantDK46763 awarded by The United States Public Health Service. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates generally to the fields of immunology andinflammatory bowel disease and more specifically to the diagnosis andtreatment of Crohn's disease using the bacterial antigen, outer membraneprotein C (OmpC).

2. Background Information

Inflammatory bowel disease (IBD) is the collective term used to describetwo gastrointestinal disorders of unknown etiology: Crohn's disease (CD)and ulcerative colitis (UC). The course and prognosis of IBD, whichoccurs world-wide and is reported to afflict as many as two millionpeople, varies widely. Onset of IBD is predominantly in young adulthoodwith diarrhea, abdominal pain, and fever the three most commonpresenting symptoms. The diarrhea may range from mild to severe, andanemia and weight loss are additional common signs of IBD. Ten percentto fifteen percent of all patients with IBD will require surgery over aten year period. In addition, patients with IBD are at increased riskfor the development of intestinal cancer. Reports of an increasedoccurrence of psychological problems, including anxiety and depression,are perhaps not surprising symptoms of what is often a debilitatingdisease that strikes people in the prime of life.

Unfortunately, the available therapies for inflammatory bowel diseaseare few, and both diagnosis and treatment have been hampered by a lackof knowledge regarding the etiology of the disease. What is clear,however, is that a combination of genetic factors, exogenous triggersand endogenous microflora can contribute to the immune-mediated damageto the intestinal mucosa seen in inflammatory bowel disease. In Crohn'sdisease, bacteria have been implicated in initiation and progression ofthe disease: the intestinal inflammation in Crohn's disease is notablefor its frequent responsiveness to antibiotics and susceptibility tobacterial fecal flow. Common intestinal colonists and novel pathogenshave been implicated in Crohn's by direct detection or by diseaseassociated anti-microbial immune responses. Furthermore, in manygenetically susceptible animal models of chronic colitis, lumenalmicro-organisms are a necessary cofactor for disease; animals housed ina germ-free environment do not develop colitis. However, despite muchdirect and indirect evidence for a role for enteric microorganisms inCrohn's disease, the pathogenic organisms and antigens contributing tothe immune dysregulation seen in this disease have not been identified.

Current diagnostic assays for Crohn's disease are unable to detect allpatients with the disease. Thus, identification of novel microbialantigens associated with Crohn's disease would provide reagents that canincrease the sensitivity of current diagnostic assays. In addition, suchmicrobial antigens can bear a disease related T-cell epitope and, asoriginal or contributing inducers of the disease-related immuneresponse, can be effective tolerogenic antigens for treating Crohn'sdisease.

Thus, there is a need for identification of microbial antigensassociated with Crohn's disease that can be used to improve thesensitivity of current diagnostic assays for this disease. The presentinvention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides a method of diagnosing Crohn's disease ina subject by determining the presence or absence of IgA anti-outermembrane protein C (OmpC) antibodies in the subject, where the presenceof the IgA anti-OmpC antibodies indicates that the subject has Crohn'sdisease. A method of the invention can be practiced, for example, byobtaining a sample from a subject; contacting the sample with an OmpCantigen, or reactive fragment thereof, under conditions suitable to forma complex of the OmpC antigen, or reactive fragment thereof, and IgAantibody to the OmpC antigen; contacting the complex with an anti-IgAantibody; and detecting the presence or absence of IgA anti-OmpCantibodies, where the presence of the IgA anti-OmpC antibodies in thesubject indicates that the subject has Crohn's disease. In oneembodiment, the invention is practiced using an OmpC antigen havingsubstantially the amino acid sequence of SEQ ID NO: 1. In anotherembodiment, the IgA anti-OmpC antibodies are detected with anenzyme-linked immunosorbent assay.

The invention further provides a method of diagnosing Crohn's disease bydetermining the presence or absence of IgA anti-OmpC antibodies in thesubject and the presence or absence of IgA anti-Saccharomyces cerevisiaeantibodies (ASCA) in the subject, where the presence of IgA anti-OmpCantibodies or the presence of IgA ASCA in the subject each independentlyindicates that the subject has Crohn's disease. The presence of IgA ASCAcan be determined, for example, by reactivity with purified yeast cellwall phosphopeptidomannan (PPM), which can be prepared, for example,from ATCC strain #38926.

The invention additionally provides a method of diagnosing Crohn'sdisease by determining the presence or absence of IgA anti-OmpCantibodies in the subject and the presence or absence of IgA anti-I-2polypeptide antibodies in the subject, where the presence of IgAanti-OmpC antibodies or the presence of IgA anti-I-2 polypeptideantibodies in the subject each independently indicates that the subjecthas Crohn's disease. In one embodiment, the presence of IgA anti-I-2polypeptide antibodies is determined by IgA reactivity against an I-2polypeptide having substantially the amino acid sequence of SEQ ID NO:3.

Further provided by the invention is a method of diagnosing Crohn'sdisease in a subject by determining the presence or absence of IgAanti-OmpC antibodies in the subject; determining the presence or absenceof IgA ASCA in the subject; and determining the presence or absence ofIgA anti-I-2 polypeptide antibodies in the subject, where the presenceof the IgA anti-OmpC antibodies, the presence of IgA ASCA or thepresence of IgA anti-I-2 polypeptide antibodies each independentlyindicates that the subject has Crohn's disease. In one embodiment, thismethod further includes the step of determining the presence or absenceof perinuclear anti-neutrophil antibodies (pANCA) in the subject.

The invention further provides a method of inducing tolerance in apatient with Crohn's disease by administering an effective dose of anOmpC antigen, or tolerogenic fragment thereof, to the patient withCrohn's disease. An OmpC antigen useful in a method of inducingtolerance can have, for example, substantially the amino acid sequenceof SEQ ID NO: 1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the IgA OmpC reactivity and IgA I-2 reactivity of sera frompatients having Crohn's disease.

FIG. 2 shows IgA OmpC reactivity and IgA ASCA reactivity of sera frompatients having Crohn's disease.

FIG. 3 shows IgA OmpC reactivity and IgG OmpC reactivity of sera frompatients having Crohn's disease.

FIG. 4 shows IgA and IgG OmpC reactivity of sera from normalindividuals.

FIG. 5 shows the E. coli OmpC amino acid sequence (SEQ ID NO: 1).

FIG. 6 shows the I-2 nucleotide sequence (SEQ ID NO: 2) and predictedamino acid sequence (SEQ ID NO: 3).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the exciting discovery that IgAantibodies to outer membrane protein C (OmpC) are associated withCrohn's disease. As shown in FIG. 4, only a single individual withoutCrohn's disease (“normal”) had IgA OmpC reactivity greater than 0.2₄₀₅,although many normals showed significant IgG OmpC reactivity. Incontrast to the normals, significant IgA OmpC reactivity was present insera from many patients having Crohn's disease (see FIG. 1).Furthermore, this reactivity was apparently distinct, in large part,from IgA I-2 and IgA ASCA reactivity seen in sera from Crohn's diseasepatients (FIGS. 1 and 2). These results are summarized in Table 1, whichindicates that IgA OmpC reactivity together with IgA ASCA reactivity,pANCA reactivity and I-2 polypeptide reactivity is a highly sensitivediagnostic system which can detect 86% of patients with Crohn's disease.Furthermore, as shown in Table 2, IgA OmpC reactivity itself detected55% of patients having Crohn's disease. These results indicate that IgAOmpC reactivity can be valuable in increasing the number of Crohn'sdisease patients that are diagnosed with the disease, therebyfacilitating earlier and more appropriate treatment.

TABLE 1 CD COHORT ASCA IgA Additional Cumulative Total Panel+ OmpC+pANCA I2 % Detected % Detected CD Cohort 153 86 56% 56% ASCA Panel - 6731 20% 76% ASCA Panel -/ 36 12  8% 84% OmpC- ASCA Panel -/ 24 2  1% 86%OmpC-/pANCA- Total % Detected: 86% 86%

TABLE 2 CD COHORT ASCA Total Panel+ OmpC+ pANCA I2 n: 153 86 84 36 79 %detected: 56% 55% 24% 52%

Based on the above, the present invention provides a method ofdiagnosing Crohn's disease in a subject by determining the presence orabsence of IgA anti-OmpC antibodies in the subject, where the presenceof the IgA anti-OmpC antibodies indicates that the subject has Crohn'sdisease. A method of the invention can be practiced, for example, byobtaining a sample from a subject; contacting the sample with an OmpCantigen, or reactive fragment thereof, under conditions suitable to forma complex of the OmpC antigen, or reactive fragment thereof, and IgAantibody to the OmpC antigen; contacting the complex with an anti-IgAantibody; and detecting the presence or absence of IgA anti-OmpCantibodies, where the presence of the IgA anti-OmpC antibodies in thesubject indicates that the subject has Crohn's disease. In oneembodiment, the invention is practiced using an OmpC antigen havingsubstantially the amino acid sequence of SEQ ID NO: 1. In anotherembodiment, the IgA anti-OmpC antibodies are detected with anenzyme-linked immunosorbent assay.

The outer-membrane protein C (“OmpC”) useful in the methods of theinvention is a “porin,” a class of transmembrane proteins that are foundin the outer membranes of bacteria, including gram-negative entericbacteria such as E. coli. The porins in the outer membrane of an E. colicell provide channels for passage of disaccharides, phosphate andsimilar molecules. Porins can be trimers of identical subunits arrangedto form a barrel-shaped structure with a pore at the center (Lodish etal., Molecular Cell Biology, Chapter 14 (1995)).

OmpC is one of the major porin proteins found in the outer membranes ofbacteria such as E. coli. OmpC is similar in structure and function toouter-membrane protein F (“OmpF”). Both assemble as trimers in the outermembrane to form aqueous channels that allow the passive diffusion ofsmall, hydrophilic molecules across the hydrophobic barrier. However,OmpC pores have a diameter of 1.1 nm, while OmpF pores have a diameterof 1.2 nm. This difference results in a slower rate of diffusion throughthe OmpC pores than through the OmpF pores.

Porin expression can be influenced by environmental conditions,including osmolarity, temperature, growth phase and toxin concentration.For example, in the intestine, where both nutrient and toxic moleculeconcentrations are relatively high, OmpC, with a smaller pore diameter,is the predominant porin (Pratt et al., Mol. Micro., 20:911–917 (1996)).

As used herein, the term “OmpC antigen” or “OmpC” means a protein thathas linear or conformational homology to OmpC. A OmpC antigen can bederived from a gram-negative bacterium, such as E. coli, and can be aspecies homolog of E. coli OmpC (SEQ ID NO: 1), which is shown in FIG.5. In nature, an OmpC antigen is a protein that forms a trimericstructure in the outer membrane of bacteria which allows the passage ofsmall molecules, or a precursor of such a protein.

As used herein, the term OmpC antigen or OmpC means a protein that hasat least 50% amino acid identity with E. coli OmpC (SEQ ID NO: 1) shownin FIG. 5. An OmpC antigen can have, for example, at least 60%, 70%,80%, 85%, 90% or 95% amino acid identity with SEQ ID NO: 1, said aminoacid identity determined with CLUSTALW using the BLOSUM 62 matrix withdefault parameters. For use in the methods of the invention, an OmpCantigen can be partially purified, for example, by spheroplast lysisfrom OmpA and OmpF deficient cells as described in Example IV, or can besimilarly prepared from a variety of other E. coli strains, which cancontain OmpA and OmpF in addition to OmpC. An OmpC antigen also can beprepared recombinantly by expressing an encoding nucleic acid sequencesuch as that available as GenBank accession K00541 by methods well knownin the art (see, for example, Ausubel et al., Current Protocols inMolecular Biology John Wiley & Sons, Inc. New York (1999)).

The methods of the invention relate to determining the presence orabsence of IgA anti-OmpC antibodies in a subject. As used herein, the“presence of IgA anti-OmpC antibodies” means IgA reactivity against anOmpC antigen that is greater than two standard deviations above the meanIgA anti-OmpC reactivity of control (normal) sera analyzed under thesame conditions.

The methods of the invention relate to the diagnosis and treatment ofCrohn's disease (regional enteritis), which is a disease of chronicinflammation that can involve any part of the gastrointestinal tract.Commonly the distal portion of the small intestine (ileum) and cecum areaffected. In other cases, the disease is confined to the smallintestine, colon or anorectal region. Crohn's disease occasionallyinvolves the duodenum and stomach, and more rarely the esophagus andoral cavity.

The variable clinical manifestations of Crohn's disease are, in part, aresult of the varying anatomic localization of the disease. The mostfrequent symptoms of Crohn's disease are abdominal pain, diarrhea andrecurrent fever. Crohn's disease is commonly associated with intestinalobstruction or fistula, which is an abnormal passage between diseasedloops of bowel, for example. Crohn's disease also includes complicationssuch as inflammation of the eye, joints and skin; liver disease; kidneystones or amyloidosis. In addition, CD is associated with an increasedrisk of intestinal cancer.

Several features are characteristic of the pathology of Crohn's disease.The inflammation associated with CD, known as transmural inflammation,involves all layers of the bowel wall. Thickening and edema, forexample, typically also appear throughout the bowel wall, with fibrosisalso present in long-standing disease. The inflammation characteristicof CD also is discontinuous in that segments of inflamed tissue, knownas “skip lesions,” are separated by apparently normal intestine.Furthermore, linear ulcerations, edema, and inflammation of theintervening tissue lead to a “cobblestone” appearance of the intestinalmucosa, which is distinctive of CD.

A hallmark of Crohn's disease is the presence of discrete aggregationsof inflammatory cells, known as granulomas, which are generally found inthe submucosa. Some Crohn's disease cases display the typical discretegranulomas, while others show a diffuse granulomatous reaction ornonspecific transmural inflammation. As a result, the presence ofdiscrete granulomas is indicative of CD, although the absence ofgranulomas also is consistent with the disease. Thus, transmural ordiscontinuous inflammation, rather than the presence of granulomas, is apreferred diagnostic indicator of Crohn's disease (Rubin and Farber,Pathology (Second Edition) Philadelphia: J.B. Lippincott Company(1994)).

In contrast to ulcerative colitis, which is characterized by acontinuous inflammation of the colon that usually is more severedistally than proximally, Crohn's disease is a patchy disease withfrequent sparing of the rectum. Furthermore, the inflammation is Crohn;sdisease is distinct from the superficial inflammation seen in ulcerativecolitis, which is usually limited to the mucosal layer and ischaracterized by an acute inflammatory infiltrate with neutrophils andcrypt abscesses. Instead, Crohn's disease affects the entire thicknessof the bowel wall with granulomas often, although not always, present.Furthermore, involvement of the terminal ileum, a cobblestone-likeappearance, discrete ulcers or fistulas suggest Crohn's disease.Characteristics that serve to distinguish Crohn's disease fromulcerative colitis are summarized in Table 3 (Rubin and Farber, supra,1994).

TABLE 3 Characteristic Features of Crohn's disease and ulcerativecolitis Feature Crohn's Disease Ulcerative Colitis Macroscopic Thickenedbowel wall Typical Uncommon Luminal narrowing Typical Uncommon “Skip”lesions Common Absent Right colon predominance Typical Absent Fissuresand fistulas Common Absent Circumscribed ulcers Common Absent Confluentlinear ulcers Common Absent Pseudopolyps Absent Common MicroscopicTransmural inflammation Typical Uncommon Submucosal fibrosis TypicalAbsent Fissures Typical Rare Granulomas Common Absent Crypt abscessesUncommon Typical

As used herein, the term “subject” means any animal capable of havinginflammatory bowel disease, including a human, non-human primate,rabbit, rat or mouse, especially a human. A subject can have one or moresymptoms of Crohn's disease or ulcerative colitis, or may beasymptomatic.

A sample useful in the methods of the invention can be obtained from anybiological fluid having antibodies such as, for example, whole blood,plasma, saliva, or other bodily fluid or tissue, preferably serum.

As used herein, the term “fragment” means a peptide, polypeptide orcompound containing naturally occurring amino acids, non-naturallyoccurring amino acids or chemically modified amino acids. A reactivefragment or tolerogenic fragment of an OmpC antigen also can be apeptide mimetic, which is a non-amino acid chemical structure thatmimics the structure of a peptide having an amino acid sequence,provided that the peptidomimetic retains preferential reactivity withIgA antibodies in sera of Crohn's disease patients or tolerogenicactivity, as defined herein. Such a mimetic generally is characterizedas exhibiting similar physical characteristics such as size, charge orhydrophobicity in the same spatial arrangement found in its peptidecounterpart. A specific example of a peptide mimetic is a compound inwhich the amide bond between one or more of the amino acids is replaced,for example, by a carbon—carbon bond or other bond well known in the art(see, for example, Sawyer, Peptide Based Drug Design, ACS, Washington(1995)).

As used herein, the term “amino acid” refers to one of the twentynaturally occurring amino acids, including, unless stated otherwise,L-amino acids and D-amino acids. The term amino acid also refers tocompounds such as chemically modified amino acids including amino acidanalogs, naturally occurring amino acids that are not usuallyincorporated into proteins such as norleucine, and chemicallysynthesized compounds having properties known in the art to becharacteristic of an amino acid, provided that the compound can besubstituted within a peptide such that it retains reactivity withCrohn's disease sera or tolerogenic activity. Examples of amino acidsand amino acids analogs are listed in Gross and Meienhofer, ThePeptides: Analysis, Synthesis, Biology, Academic Press, Inc., New York(1983). An amino acid also can be an amino acid mimetic, which is astructure that exhibits substantially the same spatial arrangement offunctional groups as an amino acid but does not necessarily have boththe α-amino and α-carboxyl groups characteristic of an amino acid.

A reactive or tolerogenic fragment of an OmpC antigen useful in theinvention can be produced or synthesized using methods well known in theart. Such methods include recombinant DNA methods and chemical synthesismethods for production of a peptide. Recombinant methods of producing apeptide through expression of a nucleic acid sequence encoding thepeptide in a suitable host cell are well known in the art and aredescribed, for example, in Ausubel et al., supra, 1999.

A reactive or tolerogenic fragment of an OmpC antigen useful in theinvention also can be produced by chemical synthesis, for example, bythe solid phase peptide synthesis method of Merrifield et al., J. Am.Chem. Soc. 85:2149 (1964). Standard solution methods well known in theart also can be used to synthesize a reactive or tolerogenic fragmentuseful in the invention (see, for example, Bodanszky, Principles ofPeptide Synthesis, Springer-Verlag, Berlin (1984) and Bodanszky, PeptideChemistry, Springer-Verlag, Berlin (1993)). A newly synthesized peptidecan be purified, for example, by high performance liquid chromatography(HPLC), and can be characterized using, for example, mass spectrometryor amino acid sequence analysis.

It is understood that limited modifications can be made to an OmpCantigen without destroying its biological function. Similarly, limitedmodifications can be made to a reactive fragment of an OmpC antigen or atolerogenic fragment of this antigen without destroying its reactivityor tolerogenic activity. A modification of an antigen disclosed hereinthat does not destroy its preferential reactivity with IgA antibodies inthe sera of patients having Crohn's disease or a modification of anantigen disclosed herein that does not destroy tolerogenic activity iswithin the definition of an OmpC antigen. Similarly, a modification of areactive fragment of an antigen disclosed herein that does not destroyits ability to form a complex with IgA antibodies in the sera of apatient having Crohn's disease is within the definition of a reactivefragment. Furthermore, a modification of a tolerogenic fragment of anOmpC antigen that does not destroy its ability to produce a decreasedimmunological response is within the definition of a tolerogenicfragment of an OmpC antigen. A modification can be, for example, anaddition, deletion, or substitution of amino acid residues; substitutionof a compound that mimics amino acid structure or function; or additionof chemical moieties such as amino or acetyl groups. The activity of amodified OmpC antigen or a modified fragment of an OmpC antigen can beassayed, for example, using one of the assays for reactivity ortolerogenic activity disclosed herein.

A particularly useful modification confers increased stability.Incorporation of one or more D-amino acids is a modification useful inincreasing stability of a OmpC antigen or fragment thereof. Similarly,deletion or substitution of lysine can increase stability by protectingagainst degradation. For example, such a substitution can increasestability and, thus, bioavailability of an OmpC antigen or a tolerogenicfragment thereof, provided that the substitution does not significantlyimpair reactivity or tolerogenic activity.

The invention further provides a method of diagnosing Crohn's disease bydetermining the presence or absence of IgA anti-OmpC antibodies in thesubject and the presence or absence of IgA anti-Saccharomyces cerevisiaeantibodies (ASCA) in the subject, where the presence of IgA anti-OmpCantibodies or the presence of IgA ASCA in the subject each independentlyindicates that the subject has Crohn's disease. The presence of IgA ASCAcan be determined, for example, by reactivity with purified yeast cellwall phosphopeptidomannan (PPM), which can be prepared, for example,from ATCC strain #38926. Methods for determining the presence of IgAASCA are exemplified herein in Example III. As used herein, the“presence of IgA ASCA” means IgA reactivity against S. cerevisiae thatis greater than 20% of the reactivity given by reference (knownpositive) Crohn's disease sera.

Anti-Saccharomyces cerevisiae antibodies (ASCA) are characteristicallyelevated in patients having Crohn's disease although the nature of theS. cerevisiae antigen supporting the specific antibody response in CD isunknown (Sendid et al., Clin. Diag. Lab. Immunol., 3:219–226 (1996)).These antibodies may represent a response against yeasts present incommon food or drink or a response against yeasts that colonize thegastrointestinal tract. Studies with periodate oxidation have shown thatthe epitopes recognized by ASCA in CD patient sera containpolysaccharides. Oligomannosidic epitopes are shared by a variety oforganisms including different yeast strains and genera, filamentousfungi, viruses, bacteria and human glycoproteins. Thus, themannose-induced antibody responses in CD may represent a responseagainst a pathogenic yeast organism or may represent a response againsta cross-reactive oligomannosidic epitope present, for example, on ahuman glycoprotein autoantigen. Regardless of the nature of the antigen,elevated levels of serum ASCA are a differential marker for Crohn'sdisease, with only low levels of ASCA reported in UC patients (Sendid etal., supra, 1996).

IgA ASCA can be detected using an antigen specific for ASCA, which isany antigen or mixture of antigens that is bound specifically by ASCA.Although ASCA antibodies were initially characterized by their abilityto bind S. cerevisiae, those of skill in the art will understand that anantigen specific for ASCA can be obtained from S. cerevisiae, or can beobtained from a variety of other sources so long as the antigen iscapable of binding specifically to ASCA antibodies. Accordingly,exemplary sources of an antigen specific for ASCA contemplated for usein the methods of the invention include whole killed yeast cells, suchas from the genera Saccharomyces and Candida, yeast cell wallphosphopeptidomannan (PPM), oligomannosides, neoglycolipids, anti-ASCAidiotypic antibodies, and the like. As described above, differentspecies and strains of yeast, including Saccharomyces, can be used as anantigen specific for ASCA in the methods provided herein. For example,S. cerevisiae strain Su1, Su2, CBS 1315 or BM 156, or Candida albicansstrain VW32, can be used as an antigen specific for ASCA in the methodsof the invention.

Preparations of yeast cell wall mannans, or phosphopeptidomannans (PPM),are also contemplated herein as antigens specific for ASCA. These watersoluble surface antigens can be prepared by appropriate extractiontechniques, including autoclaving as described in Example III or can beobtained commercially (see Lindberg et al., Gut 33:909–913 (1992)). Theacid stable fraction of yeast cell wall PPM also can be useful in themethods of the invention (Sendid et al., supra, 1996). An exemplary PPMfor use in diagnosing clinical subtypes of Crohn's disease is derivedfrom S. cerevisiae strain ATCC #38926.

Purified oligosaccharide antigens, such as oligomannosides specific forASCA, also are contemplated for use in the methods of the invention. Foruse herein, the purified oligomannoside antigens are preferablyconverted into neoglycolipids as described in Faille et al., Eur. J.Microbiol. Infect. Dis. 11:438–446 (1992). One skilled in the artunderstands that the reactivity of such an oligomannoside antigen withASCA can be optimized by varying the mannosyl chain length (Frosh etal., Proc. Natl. Cad. Sci. USA, 82:1194–1198 (1985)); the anomericconfiguration (Fukazawa et al., In E. Kurstak (ed.), Immunology ofFungal Disease, Marcel Dekker Inc., New York, pp. 37–62 (1989);Nishikawa et al, Microbiol. Immunol., 34:825–840 (1990); Poulain et al.,Eur. J. Clin. Microbiol, 23:46–52 (1993); Shibata et al., Arch. Biochem.Biophys., 243:338–348 (1985); and Trinel et al., Infect. Immun.,60:3845–3851 (1992)); or the position of the linkage (Kikuchi et al.,Planta, 190:525–535 (1993)).

An oligomannoside antigen specific for ASCA can include themannotetraose Man(1→3)Man(1→2)Man(1→2)Man, and can be purified from PMMas described in Faille et al., supra, 1992. An exemplary neoglycolipidfor use in the methods of the invention can be constructed by releasingthe oligomannoside from its respective PPM and subsequently coupling thereleased oligomannoside to 4-hexadecylaniline or the like.

The invention additionally provides a method of diagnosing Crohn'sdisease by determining the presence or absence of IgA anti-OmpCantibodies in the subject and the presence or absence of IgA anti-I-2polypeptide antibodies in the subject, where the presence of IgAanti-OmpC antibodies or the presence of IgA anti-I-2 polypeptideantibodies in the subject each independently indicates that the subjecthas Crohn's disease. In one embodiment, the presence of IgA anti-I-2polypeptide antibodies is determined by IgA reactivity against an I-2polypeptide having substantially the amino acid sequence of SEQ ID NO:3.

The methods of the invention relate to determining the presence orabsence of IgA anti-I-2 polypeptide antibodies in a subject. As usedherein, the “presence of IgA anti-I-2 polypeptide antibodies” or “IgAanti-I-2 antibodies” means IgA reactivity against an I-2 polypeptidethat is greater than two standard deviations above the IgA anti-I-2 meanreactivity of control (normal) sera analyzed under the same conditions.

As used herein, the term “I-2 polypeptide” means a polypeptide havingsubstantially the same amino acid sequence as the microbial I-2polypeptide (SEQ ID NO: 3) shown in FIG. 6. The microbial I-2polypeptide (SEQ ID NO: 3) is a polypeptide of 100 amino acids sharingsome similarity to bacterial transcriptional regulators, with thegreatest similarity in the amino-terminal 30 amino acids. The I-2encoding nucleic acid (SEQ ID NO: 2) is differentially present ininvolved Crohn's disease tissue, as compared to mucosa macroscopicallyfree of disease. An I-2 polypeptide, or reactive fragment thereof, canbe prepared, for example, using recombinant methods as set forth inExample IV.

An I-2 polypeptide having substantially the same amino acid sequence asSEQ ID NO: 3 can be the naturally occurring I-2 polypeptide (SEQ ID NO:3) or a related polypeptide having substantial amino acid sequencesimilarity to this sequence. Such related polypeptides include isotypevariants or homologs of the amino acid sequence shown in FIG. 6. As usedherein, the term I-2 polypeptide generally describes polypeptidesgenerally having an amino acid sequence with greater than about 50%identity, preferably greater than about 60% identity, more preferablygreater than about 70% identity, and can be a polypeptide having greaterthan about 80%, 90%, 95%, 97%, or 99% amino acid sequence identity withSEQ ID NO: 3, said amino acid identity determined with CLUSTALW usingthe BLOSUM 62 matrix with default parameters.

Further provided by the invention is a method of diagnosing Crohn'sdisease in a subject by determining the presence or absence of IgAanti-OmpC antibodies in the subject; determining the presence or absenceof IgA ASCA in the subject; and determining the presence or absence ofIgA anti-I-2 polypeptide antibodies in the subject, where the presenceof the IgA anti-OmpC antibodies, the presence of IgA ASCA or thepresence of IgA anti-I-2 polypeptide antibodies each independentlyindicates that the subject has Crohn's disease.

Previous studies have shown ANCA reactivity in a small portion ofpatients with Crohn's disease, although these antibodies are elevatedmore frequently in patients with ulcerative colitis. The reportedprevalence in CD varies from 0 to 43% with most studies reporting that10 to 30% of CD patients express ANCA (see, for example, Saxon et al.,J. Allergy Clin. Immunol. 86:202–210 (1990); Cambridge et al., Gut33:668–674 (1992); Pool et al., Gut 3446–50 (1993); and Brokroelofs etal., Dig. Dis. Sci. 39:545–549 (1994).

In a method of the invention, the presence or absence of pANCA can beoptionally determined in the subject, for example, by reactivity withfixed neutrophil. As used herein, the term “perinuclear anti-neutrophilcytoplasmic antibody” is synonymous with “pANCA” and refers to anantibody that reacts specifically with a neutrophil to give perinuclearto nuclear staining or cytoplasmic staining with perinuclearhighlighting. A method for determining the presence of pANCA in asubject is exemplified herein in Example V.

The invention further provides a method of inducing tolerance in apatient with Crohn's disease by administering an effective dose of anOmpC antigen, or tolerogenic fragment thereof, to the patient withCrohn's disease. An OmpC antigen useful in a method of inducingtolerance can include, for example, the sequence SEQ ID NO: 1.

As used herein, the term “effective dose” means the amount of an OmpCantigen, or a tolerogenic fragment thereof, useful for inducingtolerance in a patient having Crohn's disease. For induction of oraltolerance, for example, multiple smaller oral doses can be administeredor a large dose can be administered. Such doses can be extrapolated, forexample, from the induction of tolerance in animal models (see, forexample, Trentham et al., Science 261:1727–1730 (1993)).

Various molecules are known in the art to cause, promote or enhancetolerance, and an OmpC antigen or tolerogenic fragment thereof can becombined, if desired, with a tolerogizing molecule. See, for example,U.S. Pat. No. 5,268,454, and citations therein, each of which isincorporated herein by reference. As used herein, the term “tolerogizingmolecule” means a molecule, compound or polymer that causes, promotes orenhances tolerogenic activity when combined with an OmpC antigen orfragment thereof. A tolerogizing molecule can be optionally conjugatedto an OmpC antigen and can be, for example, polyethylene glycol. Suchmolecules are well known in the art (see, for example, U.S. Pat. No.5,268,454, supra).

An effective dose of an OmpC antigen, or a fragment thereof, forinducing tolerance can be administered by methods well known in the art.Oral tolerance is well-recognized in the art as a method of treatingautoimmune disease (see, for example, Weiner, Hospital Practice, pp.53–58 (Sep. 15, 1995)). For example, orally administered autoantigenssuppress several experimental autoimmune models in a disease- andantigen-specific fashion; the diseases include experimental autoimmuneencephalomyelitis, uveitis, and myasthenia, collagen- andadjuvant-induced arthritis, and diabetes in the NOD mouse (see, forexample, Weiner et al., Ann. Rev. Immunol. 12:809–837 (1994)).Furthermore, clinical trials of oral tolerance have produced positiveresults in treating multiple sclerosis, rheumatoid arthritis anduveitis. Modes of administration include parenteral administration andsubcutaneous injection (Johnson, Ann. Neurology 36(suppl.):S115–S117(1994)).

The term “tolerogenic fragment,” as used in reference to an OmpCantigen, means a peptide or polypeptide portion of the antigen that hastolerogenic activity as defined by its ability either alone, or incombination with another molecule, to produce a decreased immunologicalresponse. Thus, a tolerogenic fragment of an OmpC antigen is a peptideor polypeptide that has substantially the same amino acid sequence as aportion of SEQ ID NO: 1 and tolerogenic activity as defined by itsability either alone, or in combination with another molecule, toproduce a decreased immunological response. A tolerogenic fragment of anOmpC antigen can have from about three amino acids to about 90 aminoacids. A tolerogenic fragment of an OmpC antigen can have, for example,at least 5, 8, 10, 12, 15, 18, 20 or 25 amino acids. For example, atolerogenic fragment of an OmpC antigen can have from five to fiftyamino acids, from eight to fifty amino acids, or from ten to fifty aminoacids. More preferably, a tolerogenic fragment has from eight to twentyamino acids or from ten to twenty amino acids. Most preferably, atolerogenic fragment has from twelve to twenty amino acids or fromfifteen to twenty amino acids.

A tolerogenic fragment of an OmpC antigen can be identified using one ofa variety of assays, including in vitro assays such as T-cellproliferation or cytokine secretion assays and in vivo assays such asthe induction of tolerance in murine models of inflammatory boweldisease. T-cell proliferation assays, for example, are well recognizedin the art as predictive of tolerogenic activity (see, for example,Miyahara et al., Immunol. 86:110–115 (1995) or Lundin et al, J. Exp.Med. 178:187–196 (1993)). A T-cell proliferation assay can be performedby culturing T-cells with irradiated antigen-presenting cells, such asnormal spleen cells, in microtiter wells for 3 days with varyingconcentrations of the fragment to be assayed; adding ³H-thymidine; andmeasuring incorporation of ³H-thymidine into DNA. In such an assay, afragment of an OmpC antigen can be tested for activity, for example, atconcentrations of 20 μg/ml and 40 μg/ml.

A tolerogenic fragment of an OmpC antigen can be identified using aT-cell cytokine secretion assay known in the art. For example, T cellscan be cultured with irradiated antigen-presenting cells in microtiterwells with varying concentrations of the fragment of interest and, afterthree days, the culture supernatants can be assayed for IL-2, IL-4 orIFN-γ as described in Czerinsky et al., Immunol. Rev. 119:5–22 (1991).

Primary T-cells for use in a T-cell proliferation assay or cytokinesecretion assay, for example, can be isolated from lamina propria orperipheral blood. In addition, a convenient source of T-cells for use inan in vitro assay for tolerogenic activity can be a T-cell lineestablished from an IBD patient such as a Crohn's disease patient, froma murine model of IBD or from a healthy animal immunized with an OmpCantigen of the invention. A preferred source of T-cells for use inidentifying a tolerogenic fragment of an OmpC antigen is a Crohn'sdisease patient.

A T-cell line can be established from a patient with CD or UC, forexample, by culturing T lymphocytes with about 1 μg/ml recombinant OmpCantigen or OmpC fusion protein, in the presence of low concentrations ofgrowth-supporting IL-2 (about 10 μg/ml). A T-cell line can beestablished by culturing T lymphocytes with antigen-presenting cells andfeeding the cells on an alternating four to five day cycle with eitherIL-2 and an OmpC antigen, or IL-2 alone, as described in Nanda et al.,J. Exp. Med. 176:297–302 (1992). A cell line that develops into areliably proliferating cell line dependent on the presence of an OmpCantigen is particularly useful in identifying tolerogenic fragments. Theestablishment of T-cell lines from small intestinal mucosa is described,for example, in Lundin et al., supra, 1993. T cell lines dependent uponthe presence of an OmpC antigen and useful for identifying tolerogenicfragments of an OmpC antigen can be prepared similarly.

A tolerogenic fragment also can be identified by its ability to inducetolerance in vivo, as indicated by a decreased immunological response,which can be a decreased T-cell response, such as a decreasedproliferative response or cytokine secretion response as describedabove, or a decreased antibody titer to the antigen. A neonatal or adultmouse can be tolerized with a fragment of an OmpC antigen, and a T-cellresponse or anti-OmpC antibody titer can be assayed after challenging byimmunization. For example, a neonatal mouse can be tolerized within 48hours of birth by intraperitoneal administration of about 100 μg of afragment of an OmpC antigen emulsified with incomplete Freund's adjuvantand subsequently immunized with I-2 polypeptide at about 8 weeks of age(see, for example, Miyahara, supra, 1995). An adult mouse can betolerized intravenously with about 0.33 mg of a fragment of an OmpCantigen, administered daily for three days (total dose 1 mg), andimmunized one week later with an OmpC antigen. A decreased T-cellresponse, such as decreased proliferation or cytokine secretion, whichindicates tolerogenic activity, can be measured using T-cells harvested10 days after immunization. In addition, a decreased anti-OmpC antibodytiter, which also indicates tolerogenic activity, can be assayed usingblood harvested 4–8 weeks after immunization. Methods for assaying aT-cell response or anti-OmpC antigen antibody titer are described aboveand are well known in the art.

A tolerogenic fragment of an OmpC antigen also can be identified using amurine model of inflammatory bowel disease. Neonatal or adult micehaving IBD-like disease can be tolerized with a fragment of an OmpCantigen as described above, and the T-cell response or anti-OmpCantibody titer assayed. A decreased T-cell response or decreasedantibody titer to the antigen indicates a decreased immunologicalresponse and, thus, serves to identify a tolerogenic fragment of an OmpCantigen. In addition, a tolerogenic fragment of an OmpC antigen can beidentified by the ability to reduce the frequency, time of onset orseverity of colitis in a murine model of IBD.

Several well-accepted murine models of inflammatory bowel disease can beuseful in identifying a tolerogenic fragment of an OmpC antigen. Forexample, mice with targeted disruption of the gene encoding the alphasubunit of the G-protein Gi2, is a well known model exhibiting featuresof human bowel disease (Hornquist et al., J. Immunol. 158:1068–1077(1997); Rudolph et al., Nat. Genet. 10:143–150 (1995)). Mice deficientin IL-10 as described in Kühn et al., Cell 75:263–274 (1993), and micedeficient in IL-2 as described in Sadlack et al., Cell 75:253–261(1993), also have colitis-like disease and are useful in identifying atolerogenic fragment of an OmpC antigen. Furthermore, mice withmutations in T cell receptor (TCR) α, TCR β, TCR β×δ, or the class IImajor histocompatiblility complex (MHC) as described in Mombaerts etal., Cell 75:275–282 (1993), develop inflammatory bowel disease and,thus, are useful in identifying a tolerogenic fragment of an OmpCantigen. Similarly, a fragment can be assayed for tolerogenic activityin a SCID mouse reconstituted with CD45RB CD4+ T-cells, which is awell-accepted model of inflammatory bowel disease, as described inPowrie et al., Immunity 1:553–562 (1994). Additional animal models ofIBD also are well known in the art (see, for example, Podolsky, ActaGastroenterol. Belg. 60:163–165 (1997); and Bregenholt et al., APMIS105: 655–662 (1997)). Thus, a tolerogenic fragment of an OmpC antigencan be readily identified by an in vitro or in vivo assay disclosedherein or by another assay well known in the art.

A reactive or tolerogenic fragment of an OmpC antigen can be identifiedby screening a large collection, or library, of peptides of interest orrandom peptides for reactivity or tolerogenic activity. For example, apanel of peptides spanning the entire sequence of an OmpC antigen can bescreened for reactivity or tolerogenic activity as described above. Sucha panel can be a panel of 15-mer peptides spanning the sequence of theOmpC antigen (SEQ ID NO: 1), each overlapping by three or five residueshifts using the Mimotope cleavable pin technology (Cambridge ResearchBiochemicals, Wilmington, Del.), as described by Geysen et al., Science235:1184 (1987). The panel is subsequently screened for reactivity ortolerogenic activity using one of the assays described above (see, forexample, Miyahara et al., supra, 1995). A library of peptides to bescreened also can be a population of peptides related in amino acidsequence to SEQ ID NO: 1 but having one or more amino acids that differfrom SEQ ID NO: 1.

Additional peptides to be screened include, for example, tagged chemicallibraries of peptides and peptidomimetic molecules. Peptide librariesalso comprise those generated by phage display technology. Phage displaytechnology includes the expression of peptide molecules on the surfaceof phage as well as other methodologies by which a protein ligand is orcan be associated with the nucleic acid which encodes it. Methods forproduction of phage display libraries, including vectors and methods ofdiversifying the population of peptides which are expressed, are wellknown in the art (see, for example, Smith and Scott, Methods Enzymol.217:228–257 (1993); Scott and Smith, Science 249:386–390 (1990); andHuse, WO 91/07141 and WO 91/07149). These or other well known methodscan be used to produce a phage display library which can be screened,for example, with one of the disclosed assays for reactivity ortolerogenic activity. If desired, a population of peptides can beassayed for activity en masse. For example, to identify a reactivefragment of an OmpC antigen, a population of peptides can be assayed forthe ability to form a complex with patient sera containing IgA anti-OmpCantigen reactivity; the active population can be subdivided, and theassay repeated in order to isolate the reactive fragment from thepopulation.

A reactive or tolerogenic fragment of an OmpC antigen also can beidentified by screening, for example, fragments of the polypeptideproduced by chemical or proteolytic cleavage. Methods for chemical andproteolytic cleavage and for purification of the resultant proteinfragments are well known in the art (see, for example, Deutscher,Methods in Enzymology, Vol. 182, “Guide to Protein Purification,” SanDiego: Academic Press, Inc. (1990)). For example, a chemical such ascyanogen bromide or a protease such as trypsin, chymotrypsin, V8protease, endoproteinase Lys-C, endoproteinase Arg-C or endoproteinaseAsp-N can be used to produce convenient fragments of an OmpC antigenthat can be screened for reactivity or tolerogenic activity using one ofthe assays disclosed herein.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I Purification of OmpC

This example describes purification of OmpC using spheroplast lysis.

OmpF-^(/)-/OmpA-^(/)-mutant E. coli were inoculated from a glycerolstock into 10–20 ml of Luria Bertani broth supplemented with 100 μg/mlstreptomycin (LB-Strep), and cultured vigorously at 37° C. for ˜8 hoursto log phase. This starter culture was used to inoculate one liter ofLB-strep media, and the 1 L culture grown for less than 15 hours.

The cells were harvested by centrifugation (JS-4.2, 4K/15 min/4° C.). Ifnecessary, cells were washed twice with 100 ml of ice cold 20 mM Tris-ClpH 7.5. The cells were subsequently resuspended in ice cold spheroplastforming buffer (20 mM Tris-Cl pH 7.5, 20% sucrose, 0.1 M EDTA pH 8.0, 1mg/ml lysozyme), after which the resuspended cells were incubated on icefor 20 minutes to 2 hours with occasional mixing by inversion.

If required, the spheroplasts were centrifuged (JA-17, 5.5k/10 min/4°C.) and resuspended in a smaller volume of spheroplast forming buffer(SFB). The spheroplast pellet was optionally frozen prior toresuspension in order to improve lysis efficiency. Hypotonic buffer wasavoided in order to avoid bursting the spheroplasts and releasingchromosomal DNA, which significantly decreases the efficiency of lysis.

The spheroplast preparation was diluted 14-fold into ice cold 10 mMTris-Cl pH 7.5, 1 mg/ml DNase-I, and vortexed vigorously. Thepreparation was sonicated on ice 4×30 seconds at 50% power at setting 4,with a pulse “On time” of 1 second, without foaming or overheating thesample.

Cell debris was pelleted by centrifugation (JA-17, 5–10K/10 min/4° C.),and the supernatant removed and clarified by centrifugation a secondtime (10K/10 min/4° C.). The supernatant was removed without collectingany part of the pellet, and placed into ultra centrifuge tubes. Thetubes were filled to 1.5 millimeter from top with 20 mM Tris-Cl pH 7.5.

The membrane preparation was pelleted by ultra centrifugation at 100,000g (35K/1 hour/4° C. in Beckman SW 60 swing bucket rotor). The pellet wasresuspended by homogenizing into 20 mM Tris-Cl pH 7.5 using a 1 ml bluepipette tip and squirting the pellet closely before pipetting up anddown for approximately ˜10 minutes per tube.

In a 15 ml screw cap tube filled with 4 mls, the material was extractedfor 1 hour in 20 mM Tris-Cl pH 7.5 with 1% SDS, with rotation at 37° C.The preparation was transferred to ultra centrifugation tubes, and themembrane pelleted at 100,000 g (35K/1 hour/4° C. in Beckman SW 60). Thepellet was resuspended by homogenizing into 20 mM Tris-Cl pH 7.5 asbefore. The membrane preparation was optionally left at 4° C. overnight.

OmpC was extracted for 1 hour with rotation at 37° C. in 20 mM Tris-ClpH 7.5, 3% SDS, and 0.5 M NaCl (SDS will precipitate if kept below 37°C.). The material was transferred to ultra centrifugation tubes, and themembrane pelleted by centrifugation at 100,000 g (35K/1 hour/30° C. inBeckman SW 60). Lower temperatures were avoided since further coolingwill result in extracted protein salting out of solution.

The supernatant containing extracted OmpC was then dialyzed against morethan 10,000 volumes to eliminate high salt content. SDS was removed bydetergent exchange against 0.2% Triton. Triton was removed by furtherdialysis against 50 mM Tris-Cl.

Purified OmpC, which functions as a porin in its trimeric form, ischaracterized as follows when analyzed by SDS-PAGE. Electrophoresis atroom temperature resulted in a ladder of ˜100 kDa, ˜70 kDa, and ˜30 kDabands. Heating for 10–15 minutes at 65–70° C. partially dissociated thecomplex and resulted in only dimers and monomers (˜70 kDa and ˜30 kDabands). Boiling for 5 minutes resulted in monomers of 38 kDa.

EXAMPLE II ANTI-IgA OmpC ELISA Assays

This example describes an ELISA assay for direct detection of IgAanti-OmpC antibodies in patient sera.

The OmpC direct ELISA assay was performed as follows. Plates (Immulon 3,DYNEX Technologies, Chantilly, Va.) were coated overnight at 4 C with100 μl/well of OmpC prepared as described above at 0.25 μg/ml in boratebuffered saline, pH 8.5. After three washes in 0.05% Tween 20 inphosphate buffered saline (PBS), the plates were blocked with 150μl/well of 0.5% bovine serum albumin in PBS, pH 7.4 (BSA-PBS) for 30minutes at room temperature (RT). The blocking solution was thendiscarded, and 100 μl/well of serum from Crohn's disease patients,ulcerative colitis patients and normal controls diluted 1:100 was addedand incubated for 2 hours at room temperature. After washing the platesas before, alkaline phosphatase-conjugated indicator antibody (goatanti-human IgA (α-chain specific) from Jackson ImmunoResearch, WestGrove, Pa.) was added to the plates at a dilution of 1:1000 in BSA-PBS,and the plates were incubated at room temperature for 2 hours. Theplates were subsequently washed three times with 0.05% Tween 20 inphosphate buffered saline, followed by another three washes with Trisbuffered normal saline, pH 7.5. Substrate solution (1.5 mg/ml disodiumP-nitrophenol phosphate (Amresco; Solon, Ohio), 2.5 mM MgCl₂, 0.01 MTris, pH 8.6) was added at 100 μl/well, and color was allowed to developfor one hour before the plates were read at 405 nm.

IgA OmpC positive reactivity was defined as reactivity greater than twostandard deviations above the mean reactivity obtained with control(normal) sera analyzed at the same time as the test samples.

EXAMPLE III IgA ASCA ELISA Assay

This example demonstrates that the presence of IgA anti-Saccharomycescerevisiae antibodies in patient sera can be determined using an ELISAmicroplate assay.

A. Preparation of Yeast Cell Wall Mannan

Yeast cell wall mannan was prepared as follows and as described inFaille et al. Eur. J. Clin. Microbiol. Infect. Dis. 11:438–446 (1992)and in Kocourek and Ballou et al., J. Bacteriol 100:1175–1181 (1969). Alyophilized pellet of yeast Saccharomyces uvarum was obtained from theAmerican Type Culture Collection (#38926). Yeast were reconstituted in10 ml 2×YT medium, prepared according to Sambrook et al., MolecularCloning Cold Spring Harbor Laboratory Press (1989). S. uvarum were grownfor two to three days at 30° C. The terminal S. uvarum culture wasinoculated on a 2×YT agar plate and subsequently grown for two to threedays at 30° C. A single colony was used to inoculate 500 ml 2×YT media,and grown for two to three days at 30° C. Fermentation media (pH 4.5)was prepared by adding 20 gm glucose, 2 gm bacto-yeast extract, 0.25 gmMgSO₄ and 2.0 ml 28% H₃PO₄ per liter distilled water. The 500 ml culturewas used to inoculate 50 liters of fermentation media, and the culturefermented for three to four days at 37° C.

S. uvarum mannan extract was prepared by adding 50 ml 0.02 M citratebuffer (5.88 gm/l sodium citrate; pH 7.0+/−0.1) to each 100 grams ofcell paste. The cell/citrate mixture was autoclaved at 125° C. forninety minutes and allowed to cool. After centrifuging at 5000 rpm for10 minutes, the supernatant was removed and retained. The cells werethen washed with 75 ml 0.02 M citrate buffer and the cell/citratemixture again autoclaved at 125° C. for ninety minutes. The cell/citratemixture was centrifuged at 5000 rpm for 10 minutes, and the supernatantretained.

In order to precipitate copper/mannan complexes, an equal volume ofFehling's Solution was added to the combined supernatants whilestirring. The complete Fehling's solution was prepared by mixingFehling's Solution A with Fehling's Solution B in a 1:1 ratio just priorto use. The copper complexes were allowed to settle, and the liquiddecanted gently from the precipitate. The copper/mannan precipitatecomplexes were then dissolved in 6–8 ml 3N HCl per 100 grams yeastpaste.

The resulting solution was poured with vigorous stirring into 100 ml of8:1 methanol:acetic acid, and the precipitate allowed to settle forseveral hours. The supernatant was decanted and discarded; then the washprocedure was repeated until the supernatant was colorless,approximately two to three times. The precipitate was collected on ascintered glass funnel, washed with methanol and air dried overnight. Onsome occasions, the precipitate was collected by centrifugation at 5000rpm for 10 minutes before washing with methanol and air dryingovernight. The dried mannan powder was dissolved in distilled waster,using approximately 5 ml water per gram of dry mannan powder. The finalconcentration of S. uvarum cell wall mannan was approximately 30 μg/ml.

B. Preparation of S. uvarum Mannan ELISA Plates

S. uvarum cell mannan ELISA plates were saturated with antigen asfollows. Purified S. uvarum mannan prepared as described above wasdiluted to a concentration of 100 μg/ml with phosphate bufferedsaline/0.2% sodium azide (PBS-N3). Using a multi-channel pipettor, 100μl of 100 μg/ml S. uvarum mannan was added per well of a Costar 96-wellhi-binding plate (catalogue number 3590; Costar Corp., Cambridge,Mass.). The antigen was allowed to coat the plate at 4° C. for a minimumof 12 hours. Each lot of plates was compared to a previous lot beforeuse. Plates were stored at 2–8° C. for up to one month.

C. Analysis of Patient Sera

Patient sera were analyzed in duplicate for anti-IgG or anti-IgAreactivity. Microtiter plates saturated with antigen as described abovewere incubated with phosphate buffered saline/0.05% Tween-20 for 45minutes at room temperature to inhibit nonspecific antibody binding.Patient sera were subsequently added at a dilution of 1:80 for IgA andincubated for 1 hour at room temperature. Wells were washed three timeswith PBS/0.05% Tween-20. Then a 1:1000 dilution of alkalinephosphatase-conjugated goat anti-human IgA (Jackson Immunoresearch,Westgrove, Pa.) was added, and the microtiter plates incubated for 1hour at room temperature. A solution of p-nitrophenol phosphate indiethanolamine substrate buffer was added, and color development allowedto proceed for 10 minutes. Absorbance at 405 nm was analyzed using anautomated EMAX plate reader (Molecular Devices, Sunnyvale, Calif.).

Standard binding of pooled sera from patients with an establisheddiagnosis of Crohn's disease was used as a standard reference forbinding and set to be 100 ELISA units. Results with test patient serawere expressed as a percentage of the standard binding of the referenceCD sera. ASCA-positivity was defined as IgA ASCA reactivity that wasgreater than 20% of the reference CD sera.

EXAMPLE IV IgA I-2 ELISA

This example demonstrates that the I-2 polypeptide can be determinedusing ELISA analysis.

A. GST-I-2 Fusion Protein

The full-length I-2 encoding nucleic acid sequence (SEQ ID NO: 3) wascloned into the GST expression vector pGEX. After expression in E. coli,the protein was purified on a GST column. The purified protein was shownto be of the expected molecular weight by silver staining, and hadanti-GST reactivity upon western analysis.

B. ELISA Analysis

Human IgA antibodies that bind the I-2 polypeptide (SEQ ID NO: 3) weredetected by direct ELISA assays essentially as follows. Plates (Immulon3; DYNEX Technologies; Chantilly, Va.) were coated overnight at 4° C.with 100 μl/well GST-I-2 fusion polypeptide (5 μg/ml in borate bufferedsaline, pH 8.5). After three washes in 0.05% Tween 20 in phosphatebuffered saline (PBS), the plates were blocked with 150 μl/well of 0.5%bovine serum albumin in PBS, pH 7.4 (BSA-PBS) for 30 minutes at roomtemperature. The blocking solution was then replaced with 100 μl/well ofCrohn's disease or normal control serum, diluted 1:100. The plates werethen incubated for 2 hours at room temperature and washed as before.Alkaline phosphatase conjugated secondary antibody [goat anti-human IgA(α-chain specific), Jackson ImmunoResearch, West Grove, Pa.] was addedto the IgA plates at a dilution of 1:1000 in BSA-PBS. The plates wereincubated for 2 hours at room temperature before washing three timeswith 0.05% Tween 20/PBS followed by another three washes with Trisbuffered normal saline, pH 7.5. Substrate solution (1.5 mg/ml disodiumP-nitrophenol phosphate (Aresco; Solon, Ohio) in 2.5 mM MgCl₂, 0.01 MTris, pH 8.6) was added at 100 μl/well, and color allowed to develop forone hour. The plates were then analyzed at 405 nm.

IgA I-2 positive reactivity was defined as reactivity greater than twostandard deviations above the mean reactivity obtained with control(normal) sera analyzed at the same time as the test samples.

EXAMPLE V ELISA and Indirect Immunofluorescence for Determining pANCAStatus

This example describes methods for determining the pANCA status of asubject.

A. Presence of ANCA was Determined by Fixed Neutrophil ELISA

A fixed neutrophil enzyme-linked immunosorbent assay was used to detectANCA as described in Saxon et al., J. Allergy Clin. Immunol. 86:202–210(1990), and all samples were analyzed in a blinded fashion. Microtiterplates were coated with 2.5×10⁵ neutrophils per well and treated with100% methanol to fix the cells. Cells were incubated with 0.25% bovineserum albumin (BSA) in phosphate-buffered saline to block nonspecificantibody binding. Next, control and coded sera were added at a 1:100dilution to the bovine serum/phosphate-buffered saline blocking buffer.Alkaline phosphatase conjugated goat F(ab′)₂ anti-human immunoglobulin G(γ-chain specific) antibody (Jackson Immunoresearch Labs, Inc., WestGrove, Pa.) was added at a 1:1000 dilution to label neutrophil boundantibody. A p-nitrophenol phosphate substrate solution was added andcolor development was allowed to proceed until absorbance at 405 nm inthe positive control wells was 0.8–1.0 optical density units greaterthan the absorbance in blank wells.

B. Indirect Immunofluorescence Assay for Determination of ANCA StainingPattern

Indirect immunofluorescent staining was performed on samples that wereANCA-positive by ELISA to determine whether the predominant stainingpattern was perinuclear (PANCA) or cytoplasmic (cANCA). Glass slidescontaining approximately 100,000 neutrophils per slide were prepared bycytocentrifugation (Shandon Cytospin, Cheshire, England) and they werefixed in 100% methanol, air-dried, and stored at −20° C. The fixedneutrophils were incubated with human sera were diluted (1:20), and thereaction was visualized with fluorescein-labeled F(ab′)₂ γchain-specific antibody as described in Saxon et al., supra, 1990. Theslides were examined using an epifluorescence-equipped Olympus BH-2microscope (Olympus, Lake Success, N.Y.).

pANCA positivity was defined as a perinuclear staining pattern combinedwith ELISA reactivity greater than two standard deviations above themean reactivity obtained with control (normal) sera analyzed at the sametime as the test samples.

All journal article, reference, and patent citations provided above, inparentheses or otherwise, whether previously stated or note, areincorporated herein by reference.

Although the invention has been described with reference to the examplesabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention. Accordingly, theinvention is limited only by the following claims.

1. A method of diagnosing Crohn's disease in a subject, comprisingdetermining the presence or absence of IgA anti-outer membrane protein C(anti-OmpC) antibodies in said subject, where the presence of said IgAanti-OmpC antibodies indicates that said subject has Crohn's disease. 2.A method of diagnosing Crohn's disease in a subject, comprising thesteps of: (a) obtaining a sample from a subject suspected of havinginflammatory bowel disease; (b) contacting the sample with an OmpCantigen, or reactive fragment thereof, under conditions suitable to forma complex of the OmpC antigen, or reactive fragment thereof, and IgAanti-OmpC antibody; (c) contacting said complex with a labeled anti-IgAantibody to form a labeled complex; and (d) detecting the presence orabsence of said labeled complex, thereby determining the presence orabsence of IgA anti-OmpC antibodies, where the presence of said IgAanti-OmpC antibodies in said subject indicates that said subject hasCrohn's disease.
 3. A method of diagnosing Crohn's disease in a subject,comprising the steps of: (a) contacting a sample from a subjectsuspected of having inflammatory bowel disease with an OmpC antigen, orreactive fragment thereof, under conditions suitable to form a complexof the OmpC antigen, or reactive fragment thereof, and IgA anti-OmpCantibody, wherein said OmpC antigen comprises the amino acid sequence ofSEQ ID NO:1; (b) contacting said complex with a labeled anti-IgAantibody to form a labeled complex; and (c) detecting the presence orabsence of said labeled complex, thereby determining the presence orabsence of IgA anti-OmpC antibodies, where the presence of said IgAanti-OmpC antibodies in said subject indicates that said subject hasCrohn's disease.
 4. The method of claim 2, wherein IgA anti-OmpCantibodies are detected with an enzyme-linked immunosorbent assay. 5.The method of claim 2, further comprising determining the presence orabsence of IgA anti-Saccharomyces cerevisiae antibodies (ASCA) in saidsubject, wherein the presence of IgA anti-OmpC antibodies or thepresence of IgA ASCA in said subject each independently indicates thatsaid subject has Crohn's disease.
 6. The method of claim 5, wherein thepresence of IgA ASCA is determined by reactivity with purified yeastcell wall phosphopeptidomanan (PPM).
 7. The method of claim 6, whereinsaid yeast cell wall PPM is prepared from ATCC strain #38926.
 8. Amethod of increasing the sensitivity of diagnosing Crohn's disease in asubject, comprising determining the presence or absence of IgA anti-OmpCantibodies in said subject, where the presence of said IgA anti-OmpCantibodies indicates that said subject has Crohn's disease.
 9. A methodof diagnosing Crohn's disease in a subject, comprising determining thepresence or absence of IgA anti-OmpC antibodies and the presence orabsence of IgA ASCA in said subject, where the presence of said IgAanti-OmpC antibodies and the presence of said IgA ASCA eachindependently indicate that said subject has Crohn's disease.
 10. Amethod of diagnosing Crohn's disease in a subject, comprising the stepsof: (a) obtaining a sample from a subject suspected of havinginflammatory bowel disease; (b) contacting a portion of the sample withan OmpC antigen, or reactive fragment thereof, under conditions suitableto form a complex of the OmpC antigen, or reactive fragment thereof, andIgA anti-OmpC antibody; (c) contacting said complex from step (b) with alabeled anti-IgA antibody to form a labeled complex; (d) detecting thepresence or absence of said labeled complex from step (c), therebydetermining the presence or absence of IgA anti-OmpC antibodies, (e)contacting another portion of the sample with one or more antigensspecific for IgA ASCA under conditions suitable to form a complex of theone or more antigens specific for ASCA and IgA ASCA; (f) contacting saidcomplex from step (e) with a labeled anti-IgA antibody to form a labeledcomplex; and (g) detecting the presence or absence of said labeledcomplex from step (f), thereby determining the presence or absence ofASCA, where the presence of said IgA anti-OmpC antibodies and thepresence of ASCA in said subject each independently indicate that saidsubject has Crohn's disease.
 11. The method of claim 8, wherein thepresence or absence of IgA anti-OmpC antibodies is detected incombination with detecting the presence or absence of IgA antibodiesagainst one or more additional microbial antigens.
 12. The method ofclaim 11, wherein said one or more additional microbial antigenscomprises IgA ASCA.
 13. The method of any one of claims 8, 9 or 10,wherein IgA anti-OmpC antibodies and ASCA are detected with independentenzyme-linked immunosorbent assays.
 14. The method of any one of claims8, 9 or 10, wherein said OmpC antigen comprises the amino acid sequenceof SEQ ID NO:1.
 15. The method of any one of claims 8, 9 or 10, whereinthe presence of IgA ASCA is determined by reactivity with purified yeastcell wall phosphopeptidomanan (PPM).
 16. A method of diagnosing Crohn'sdisease in a subject, comprising determining the presence or absence ofIgA anti-OmpC antibodies, IgA ASCA, IgA anti I-2 polypeptide antibodiesand peri-nuclear anti-neutrophil antibodies (pANCA) in said subject,where the presence of said IgA anti-OmpC antibodies, IgA ASCA, IgA antiI-2 polypeptide antibodies and peri-nuclear anti-neutrophil antibodies(pANCA) in said subject indicates that said subject has Crohn's disease.